Synthesis of phosphate derivatives

ABSTRACT

The present invention is a process for the preparation of certain 5′-phosphoramidate nucleotide diastereoisomers. The phosphoramidates include those useful in the treatment of cancer such as NUC-3373 (5-fluoro-2′-deoxyuridine-5′-O-[1-naphthyl(benzyloxy-L-alaninyl)]phosphate).

RELATED APPLICATIONS

This application is a § 371 national stage application based on PatentCooperation Treaty Application serial number PCT/GB2018/051638, filedJun. 14, 2018; which claims the benefit of priority to GB 1709471.5,filed Jun. 14, 2017.

FIELD OF THE INVENTION

The present invention generally relates to a novel process for thepreparation of certain ProTides as particular phosphatediastereoisomers. The certain ProTides include those useful in thetreatment of cancer such as NUC-3373(5-fluoro-2′-deoxyuridine-5′-O-[1-naphthyl(benzyloxy-L-alaninyl)]phosphate), NUC-7738(3′-deoxyadenosine-5′-O-[phenyl(benzyloxy-L-alaninyl)] phosphate) andNUC-9701 (8-chloroadenosine-5′-O-[naphthyl(benzyloxy-L-alaninyl)]phosphate)

BACKGROUND OF THE INVENTION

ProTides are masked phosphate derivatives of nucleosides. They have beenshown to be particularly potent therapeutic agents in the fields of bothantivirals and oncology. ProTides, more specifically, are prodrugs ofmonophosphorylated nucleosides. These compounds appear to avoid many ofthe inherent and acquired resistance mechanisms which limit the utilityof the parent nucleosides (see, for example, ‘Application of Pro TideTechnology to Gemcitabine: A Successful Approach to Overcome the KeyCancer Resistance Mechanisms Leads to a New Agent (NUC-1031) in ClinicalDevelopment’; Slusarczyk et al; J. Med. Chem.; 2014, 57, 1531-1542).

NUC-3373(5-fluoro-2′-deoxyuridine-5′-O-[1-naphthyl(benzoxy-L-alaninyl)]phosphate)is a ProTide adaptation of 5FU/FUDR, the current foundation treatmentagainst colorectal cancer. NUC-3373 and a range of related compoundshave shown activity in vitro against a range of cancer models, in manycases and in particular for NUC-3373 that activity was outstanding andfar superior to the results obtained with 5-fluorouracil. The additionof the ProTide phosphoramidate moiety to the 5-fluorouracil/FUDRmolecule confers the specific advantages of delivering the key activatedform of the agent (FdUMP) into the tumour cells. Non clinical studieshave demonstrated that NUC-3373 overcomes the key cancer cell resistancemechanisms associated with 5-FU and its oral pro-drug capecitabine,generating high intracellular levels of the active FdUMP metabolite,resulting in a much greater inhibition of tumour cell growth.Furthermore, in formal dog toxicology studies, NUC-3373 is significantlybetter tolerated than 5-FU (see WO2012/117246; McGuigan et al.;Phosphoramidate Pro Tides of the anticancer agent FUDR successfullydeliver the preformed bioactive monophosphate in cells and conferadvantage over the parent nucleoside; J. Med. Chem.; 2011, 54,7247-7258; and Vande Voorde et al.; The cytostatic activity of NUC-3073,a phosphoramidate prodrug of 5-fluoro-2′-deoxyuridine, is independent ofactivation by thymidine kinase and insensitive to degradation byphosphorolytic enzymes; Biochem. Pharmacol.; 2011, 82, 441-452).

ProTide derivatives of purine nucleosides such as 8-chloroadenosine and3′-deoxyadenosine and related compounds have also shown excellentactivity in vitro against a range of solid tumours, leukaemias andlymphomas (see WO2016/083830 and GB1609602.6). 3′-Deoxyadenosine itselfis not a particularly potent anticancer agent.

ProTides are typically prepared as a mixture of two diastereoisomers,epimeric at the phosphate centre. The diastereoisomers of NUC-3373, forexample, have the following structures (in which Np is a 1-napthyl):

WO 2014/076490 discloses a process for preparation of nucleosideprodrugs such as gemcitabine-[phenyl(benzoxy-L-alaninyl)] phosphate byreacting gemcitabine or its structural variants with a diastereoisomericmixture of phosphorochloridates in the presence of a catalyst comprisingmetal salt such as Cu(OTf)₂, CuCl, CuBr, Cul, Cu(OAc)₂, CuSO₄,Cu(OC(O)CF₃)₂, Cu(OTf)₂, Yb(OTf)₃, Fe(OTf)₃, La(OTf)₃ with yield of˜45%.

A method for synthesizing NUC-1031 in diastereoisomerically pure form isdescribed in WO2017/098252 (PCT/GB2016/053875).

It is an aim of certain embodiments of this invention to provide amethod of providing NUC-3373, NUC-7738 and/or NUC-9701 in substantiallydiastereoisomerically pure form.

It is an aim of certain embodiments of this invention to provide amethod of providing the (S_(p)) and/or (R_(p))-epimer(s) of NUC-3373,NUC-7738 and/or NUC-9701 in substantially diastereoisomerically pureform(s) which is scalable, economic and/or efficient, e.g. morescalable, economic and/or efficient than methods using HPLC. Thus, it isan aim of certain embodiments of this invention to provide a method ofproviding the (S_(p)) and/or (R_(p))-epimer(s) in substantiallydiastereoisomerically pure form(s) which is suitable for large scalemanufacture.

It is an aim of certain embodiments of this invention to provide asimple method i.e. a method which involves a minimum number of processsteps and or reagents of providing the (S_(p)) and/or (R_(p))-epimer(s)in substantially diastereoisomerically pure form(s).

Another aim of certain embodiments of this invention is to provide amethod which ensures the separated (S_(p))- or (R_(p))-epimer areprovided in substantially diastereoisomerically pure form and at thesame time meet or exceed the necessary criteria stipulated byorganisations such as the US FDA concerning the amounts and nature ofany trace impurities which arise from synthesis and separation.

Certain embodiments of this invention satisfy some or all of the aboveaims.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the invention there is provided aprocess for the preparation of NUC-3373 (Formula Ia) in substantiallydiastereoisomerically pure form:

the process comprising step a) and optionally step b):

-   -   a) reacting a compound of Formula IIa; wherein R¹ represents an        electron withdrawing group and a is an integer from 1 to 5, with        a compound of Formula IIIa in presence of a base (B1) to provide        a compound of Formula IVa in substantially diastereomerically        pure form; wherein P¹ is independently selected from hydrogen        and a protecting group; and wherein the compound of formula IIa        is in substantially diastereomerically pure form:

-   -   b) where P¹ is a protecting group, optionally removing the        protecting group P¹ from the compound of formula IVa to provide        NUC-3373 in substantially diastereomerically pure form.

In accordance with a second aspect of the invention there is provided aprocess for the preparation of NUC-7738 (Formula Ib) in substantiallydiastereoisomerically pure form:

the process comprising step a) and optionally step b):

-   -   a) reacting a compound of Formula IIb; wherein R¹ represents an        electron withdrawing group and a is an integer from 1 to 5, with        a compound of Formula IIIb in presence of a base (B1) to provide        a compound of Formula IVb in substantially diastereomerically        pure form; wherein P², P³ and P⁴ are each independently selected        from hydrogen and a protecting group; and wherein the compound        of formula lib is in substantially diastereomerically pure form:

-   -   b) where any one or more of P², P³ and P⁴ are protecting groups,        optionally removing the protecting groups P², P³ and P⁴ from the        compound of formula IVb to provide NUC-7738 in substantially        diastereomerically pure form.

In accordance with a third aspect of the invention there is provided aprocess for the preparation of NUC-9701 (Formula Ic) in substantiallydiastereoisomerically pure form:

the process comprising step a) and optionally step b):

-   -   a) reacting a compound of Formula IIa; wherein R¹ represents an        electron withdrawing group and a is an integer from 1 to 5, with        a compound of Formula IIIc in presence of a base (B1) to provide        a compound of Formula IVc in substantially diastereomerically        pure form; wherein P⁵, P⁶, P⁷ and P⁸ are each independently        selected from hydrogen and a protecting group; and wherein the        compound of formula IIa is in substantially diastereomerically        pure form:

-   -   b) where any one or more of P⁵, P⁶, P⁷ and P⁸ are protecting        groups, optionally removing the protecting groups P⁵, P⁶, P⁷ and        P⁸ from the compound of formula IVc to provide NUC-9701 in        substantially diastereomerically pure form.

R¹ may be selected from the group comprising: halo group (e.g. selectedfrom fluoro, bromo, chloro or iodo); trifluoromethyl, cyano and nitro. ais an integer between 1 and 5. R¹ may be at each occurrence halo, e.g.fluoro. a may be 5.

Displacement of the substituted phenoxy group takes place selectivelywith inversion of phosphate stereocentre.

Typically the (S_(p))-diastereoisomer of the precursor (the compound offormula IIa or IIb) provides the (S_(p))-diastereoisomer of the ProTideand the (R_(p))-diastereoisomer of the precursor provides the(R_(p))-diastereoisomer of the ProTide. The exception to this is whenthe OPh(R¹)_(a) leaving group has lower priority assignment under theCahn-Ingold-Prelog rules than the naphthyl group (e.g. where OPh(R¹)_(a)is paranitrophenoxy). In such cases, the (R_(p))-diastereoisomer of theprecursor (the compound of formula IIa) provides the(S_(p))-diastereoisomer of the ProTide and the (S_(p))-diastereoisomerof the precursor provides the (R_(p))-diastereoisomer of the protide.Throughout this specification, the isomer of the compound of formula IIathat provides the (S_(p))-isomer of the ProTide is referred to as theX-diastereoisomer and the isomer of the compound of formula IIa thatprovides the (R_(p))-isomer of the ProTide is referred to as theY-diastereoisomer. For compound IIb, it is always the case that the(S_(p))-diastereoisomer of the precursor (the compound of formula IIb)provides the (S_(p))-diastereoisomer of the ProTide and the(R_(p))-diastereoisomer of the precursor provides the(R_(p))-diastereoisomer of the ProTide

Thus, it may be that the process of the first, second or third aspect isa method of making the (S_(p))-diastereoisomer of the ProTide indiastereomerically enriched form and the compound of formula IIa or IIbis in diastereomerically enriched form.

It may be that the process of the first, second or third aspect is amethod of making the (R_(p))-diastereoisomer of the ProTide indiastereomerically enriched form and the compound of formula IIa or IIbis in diastereomerically enriched form.

The base (B1) might be a nitrogen base. Nitrogen bases includeN-alkylimidazoles, (e.g. N-methyl imidazole (NMI)), imidazole,optionally substituted pyridines, (e.g. collidine, pyridine,2,6-lutidine) and trialkylamines (e.g. triethylamine, anddiisopropylethylamine). Alternatively, the base (B1) may be anorganometallic base or metal hydride base (e.g. NaH). Thus, the base maybe a Grignard reagent (i.e. an alkylmagnesium halide). ExemplaryGrignard reagents include t-butylmagnesium halides such as tBuMgCl,tBuMgBr. Preferably, the base is tBuMgCl.

Step a) may be carried out in a solvent S1.

In a fourth aspect of the invention, there is provided a process for thediastereoisomeric enrichment of a compound of Formula IIa; the processcomprising:

-   -   c) suspending or dissolving the X-diastereoisomer of the        compound of Formula IIa or a mixture of the XR- and        Y-diastereoisomers of the compound of Formula IIa in a solvent        (S2),    -   d) treating the solution or suspension with a base (B2) to        obtain the X-diastereoisomer in substantially diastereomerically        enriched form, and    -   e) isolating the X-diastereoisomer of Formula IIa. Typically,        the X-diastereoisomer is the (S)-diastereoisomer and the        Y-diastereoisomer is the (R)-diastereoisomer.

The inventors have surprisingly found that upon treating compounds offormula IIa with a base, they isomerise, preferentially forming theX-diastereoisomer over the Y-diastereoisomer. Thus, theY-diastereoisomer can be converted to the X-diastereoisomer or anepimeric mixture of the Y-diastereoisomer and the X-diastereoisomer canbe converted to the X-diastereoisomer. This increases the net efficiencyof any synthetic sequence for making the X-diastereoisomer of NUC-3373or NUC-9701 which incorporates the process of the first or third aspectas it means that all of the compound of formula IIa, even a portion ofthat which originally formed as the Y-diastereoisomer can be used.Typically, the X-diastereoisomer is the (Sp)-diastereoisomer and theY-diastereoisomer is the (Rp)-diastereoisomer.

It may be that the process comprises:

-   -   forming the compound of Formula IIa as a mixture of the Y- and        X-diastereoisomers; and that step c) comprises suspending or        dissolving the mixture of the Y- and X-diastereoisomers of the        compound of Formula IIa in a solvent (S2). Typically, the        X-diastereoisomer is the (Sp)-diastereoisomer and the        Y-diastereoisomer is the (Rp)-diastereoisomer.

The compound of formula IIa used in the process of the first or thirdaspect may be X-diastereoisomer formed according to the process of thefourth aspect. Typically, the X-diastereoisomer is the(S_(p))-diastereoisomer and the Y-diastereoisomer is the(R_(p))-diastereoisomer.

The process of the second aspect of the invention may comprise:

-   -   c) suspending or dissolving the R_(p)-diastereoisomer of the        compound of Formula IIb or a mixture of the (R_(p))- and        (S_(p))-diastereoisomers of the compound of Formula IIb in a        solvent (S2),    -   d) treating the solution or suspension with a base (B2) to        obtain (S_(p))-diastereoisomer in substantially        diastereomerically enriched form, and    -   e) isolating the (S_(p))-diastereoisomer of Formula IIb.

Thus, the process of the second aspect of the invention may comprise:

-   -   forming the compound of Formula IIb as a mixture of the (R_(p))-        and (S_(p))-diastereoisomers; and that step c) comprises        suspending or dissolving the mixture of the (R_(p))- and        (S_(p))-diastereoisomers of the compound of Formula IIb in a        solvent (S2).

The base (B2) may be selected from the group consisting of organic aminebases (e.g. primary, secondary, tertiary amines, cyclic amine; exemplaryorganic amine bases include bases include N-alkylimidazoles, [e.g.N-methyl imidazole (NMI), imidazole, optionally substituted pyridines,(e.g. collidine, pyridine, 2,6-lutidine) and trialkylamines (e.g.triethylamine, and diisopropylethylamine)]; or inorganic bases (e.g.alkali metal hydroxide, alkali metal carbonates, alkali metal alkoxides,alkali metal aryloxides). Preferably, B2 is a tertiary amine. Thus, B2may be a trialkylamine. Most preferably, B2 is triethylamine.

The solvent S2 may be selected from the group consisting of amides,ethers, esters, ketones, aromatic hydrocarbons, halogenated solvents,nitriles, sulfoxides, sulfones and mixtures thereof. S2 may be anorganic solvent. Organic solvents include but are not limited to ethers(e.g. tetrahydrofuran, dioxane, diethyl ether, t-butylmethylether);ketones (e.g. acetone and methyl isobutyl ketone); halogenated solvents(e.g. dichloromethane, chloroform and 1,2-dichloroethane); hydrocarbons(e.g. cyclohexane, pentane, hexane, heptane), aromatic solvents (e.g.benzene and toluene), esters (e.g. ethyl acetate) and amides (e.g. DMF,NMP); or mixtures thereof. Preferably, S2 is a hydrocarbon or is amixture comprising a hydrocarbon. Where S2 is a mixture, it may be amixture that comprises over 50% (e.g. over 70%) of the hydrocarbon. Thehydrocarbon may be hexane. The hydrocarbon may be heptane. S2 may be amixture of hexane or heptane and a polar organic solvent (e.g. an ether,ester, alcohol or halogenated solvent). S2 may be a mixture of hexane orheptane and a polar organic solvent, the mixture comprising over 50%(e.g. over 70%) by volume hexane or heptane. S2 may be a mixture ofhexane or heptane and ethyl acetate. S2 may be a mixture of heptane andethyl acetate. S2 may be a mixture of hexane or heptane and ethylacetate, the mixture that comprising over 50% (e.g. over 70%) by volumehexane or heptane. S2 may be a mixture of heptane and ethyl acetate, themixture comprising over 50% (e.g. over 70%) by volume heptane. S2 may bea mixture of hexane or heptane and methyl-t-butylether. S2 may be amixture of hexane and methyl-t-butylether. S2 may be a mixture of hexaneor heptane and methyl-t-butylether, the mixture that comprising over 50%(e.g. over 70%) by volume hexane or heptane. S2 may be a mixture ofhexane and methyl-t-butylether, the mixture comprising over 50% (e.g.over 70%) by volume hexane.

Step d) may involve stirring the mixture of the compound of formula IIaand the base B2 for 24 h or longer. Step d) may involve stirring themixture of the compound of formula IIa and the base B2 for 48 h orlonger. Step b) may involve stirring the mixture of the compound offormula IIa and the base B2 for 60 h or longer. Step d) may involvestirring the mixture of the compound of formula IIa and the base B2 for72 h or longer. Step d) may involve stirring the mixture of the compoundof formula IIa and the base B2 for up to 100 h.

Step d) may involve stirring the mixture of the compound of formula IIaand the base B2 at a temperature from 0 to 60° C. Step d) may involvestirring the mixture of the compound of formula IIa and the base B2 at atemperature from 20 to 40° C.

Step d) may involve stirring the mixture of the compound of formula IIband the base B2 for 2 h or longer. Step d) may involve stirring themixture of the compound of formula lib and the base B2 for 6 h orlonger. Step b) may involve stirring the mixture of the compound offormula lib and the base B2 for 10 h or longer. Step d) may involvestirring the mixture of the compound of formula lib and the base B2 for16 h or longer. Step d) may involve stirring the mixture of the compoundof formula lib and the base B2 for up to 36 h.

Step d) may involve stirring the mixture of the compound of formula liband the base B2 at a temperature from 0 to 50° C. Step d) may involvestirring the mixture of the compound of formula lib and the base B2 at atemperature from 10 to 35° C.

In certain specific embodiments of the second aspect of the invention,the compound of Formula IIb is a compound selected from:

The compound of formula IIb may be:

The compound of formula IIb may be:

In certain specific embodiments of the first, third and fourth aspect ofthe invention, the compound of Formula IIa is a compound selected from:

The compound of formula IIa may be compound 12:

The compound of formula IIa may be (R_(p))-compound 12 in substantiallydiastereomerically pure form. The compound may be the fast elutingisomer of compound 12 in substantially diastereoisomerically pure form.Thus, the compound may be the isomer of compound 12 that has a ³¹P NMRpeak at −1.41±0.02 when the NMR spectrum has been obtained on a 202 MHzNMR machine in CDCl₃, said isomer being in substantiallydiastereoisomerically pure form. The compound may be the isomer ofcompound 12 that has a retention time of 12.96±0.20 minutes whenanalytical HPLC is performed on a Varian Pursuit XRs 5 C18, 150×4.6 mmeluting with H₂O/MeOH in 20/80 in 35 min at 1 mL/min, said isomer beingin substantially diastereoisomerically pure form.

The compound of formula IIa may be (S_(p))-compound 12 in substantiallydiastereomerically pure form. The compound may be the slow elutingisomer of compound 12 in substantially diastereoisomerically pure form.Thus, the compound may be the isomer of compound 12 that has a ³¹P NMRpeak at −1.36±0.02 when the NMR spectrum has been obtained on a 202 MHzNMR machine in CDCl₃, said isomer being in substantiallydiastereoisomerically pure form. The compound may be the isomer ofcompound 12 that has a retention time of 14.48±0.20 minutes whenanalytical HPLC is performed on a Varian Pursuit XRs 5 C18, 150×4.6 mmeluting with H₂O/MeOH in 20/80 in 35 min at 1 mL/min, said isomer beingin substantially diastereoisomerically pure form.

The compound of formula IIa may be:

The NUC-3373 may be the fast eluting isomer of NUC-3373 in substantiallydiastereoisomerically pure form. Thus, the NUC-3373 may be the isomer ofNUC-3373 that has a ³¹P NMR peak at 4.27±0.10 when the NMR spectrum hasbeen obtained on a 202 MHz NMR machine in CD₃OD, said isomer being insubstantially diastereoisomerically pure form. The NUC-3373 may be theisomer of NUC-3373 that has a retention time of 16.03±0.20 minutes whenanalytical HPLC is performed on a Varian Pursuit XRs 5 C18, 150×4.6 mmeluting with H₂O/CH₃CN from 100/10 to 0/100 in 35 min at 1 mL/min, saidisomer being in substantially diastereoisomerically pure form.

The NUC-3373 may be the slow eluting isomer of NUC-3373 in substantiallydiastereoisomerically pure form. Thus, the NUC-3373 may be the isomer ofNUC-3373 that has a ³¹P NMR peak at 4.62±0.10 when the NMR spectrum hasbeen obtained on a 202 MHz NMR machine in CD₃OD, said isomer being insubstantially diastereoisomerically pure form. The NUC-3373 may be theisomer of NUC-3373 that has a retention time of 16.61±0.20 minutes whenanalytical HPLC is performed on a Varian Pursuit XRs 5 C18, 150×4.6 mmeluting with H₂O/CH₃CN from 90/10 to 0/100 in 35 min at 1 mL/min, saidisomer being in substantially diastereoisomerically pure form.

The NUC-9701 may be the fast eluting isomer of NUC-9701 in substantiallydiastereoisomerically pure form. Thus, the NUC-9701 may be the isomer ofNUC-9701 that has a ³¹P NMR peak at 3.93±0.04 when the NMR spectrum hasbeen obtained on a 202 MHz NMR machine in CD₃OD, said isomer being insubstantially diastereoisomerically pure form. The NUC-9701 may be theisomer of NUC-9701 that has a retention time of 16.43±0.10 minutes whenanalytical HPLC is performed on a Varian Pursuit XRs 5 C18, 150×4.6 mmeluting with H₂O/CH₃CN from 90/10 to 0/100 in 30 min at 1 mL/min, saidisomer being in substantially diastereoisomerically pure form.

The NUC-9701 may be the slow eluting isomer of NUC-9701 in substantiallydiastereoisomerically pure form. Thus, the NUC-9701 may be the isomer ofNUC-9701 that has a ³¹P NMR peak at 3.83±0.04 when the NMR spectrum hasbeen obtained on a 202 MHz NMR machine in CD₃OD, said isomer being insubstantially diastereoisomerically pure form. The NUC-9701 may be theisomer of NUC-9701 that has a retention time of 16.59±0.10 minutes whenanalytical HPLC is performed on a Varian Pursuit XRs 5 C18, 150×4.6 mmeluting with H₂O/CH₃CN from 100/10 to 0/100 in 30 min at 1 mL/min, saidisomer being in substantially diastereoisomerically pure form.

The compound of formula IIa may be prepared according to the fourthaspect of the invention.

In a fifth aspect of the invention is provided a compound of formulaIIa. The compound may be the Sp isomer of a compound of formula IIa. Thecompound may be the Rp isomer of a compound of formula IIa.

In a sixth aspect of the invention is provided (S_(p))-NUC-3373:

in substantially diastereoisomerically pure form. The preferentialisomerization to form the X-diastereoisomer of the compound of formulaIIa, means that the S_(p) isomer of NUC-3373 is easier to produce thanthe R_(p) isomer.

In a seventh aspect of the invention is provided (R_(p))-NUC-3373:

in substantially diastereoisomerically pure form.

In an eighth aspect of the invention is provided (S_(p))-NUC-7738:

in substantially diastereoisomerically pure form. The preferentialisomerization to form the (S)-diastereoisomer of the compound of formulalib, means that the S_(p) isomer of NUC-7738 is easier to produce thanthe R_(p) isomer.

In an ninth aspect of the invention is provided (R_(p))-NUC-7738:

in substantially diastereoisomerically pure form.

In a tenth aspect of the invention is provided (S_(p))-NUC-9701:

in substantially diastereoisomerically pure form. The preferentialisomerization to form the X-diastereoisomer of the compound of formulaIIa, means that the S_(p) isomer of NUC-9701 is easier to produce thanthe R_(p) isomer.

In an eleventh aspect of the invention is provided (R_(p))-NUC-9701:

in substantially diastereoisomerically pure form.

The compound of the fifth, sixth, seventh, eighth, ninth, tenth andeleventh aspects of the invention may be a diastereoisomer describedabove for the first, second and third aspects of the invention.

The invention may also provide a pharmaceutical composition comprising acompound of the sixth, seventh, eighth, ninth, tenth and eleventhaspects of the invention and a pharmaceutically acceptable excipient.

The invention may also provide a method of treating cancer (e.g. a solidtumour or leukaemia), the method comprising administering to a subjectin need thereof a therapeutically effective amount of a compound of thesixth, seventh, eighth, ninth, tenth and eleventh aspects of theinvention.

The compounds of the sixth, seventh, eighth, ninth, tenth and eleventhaspects of the invention may be for medical use. The compounds of thesixth, seventh, eighth, ninth, tenth and eleventh aspects of theinvention may be for use in treating cancer (e.g. a solid tumour orleukaemia).

The products of the sixth, seventh, eighth, ninth, tenth and eleventhaspects of the invention may be obtainable by (or obtained by) thefirst, second or third aspects of the invention.

A protecting group for a hydroxyl group (e.g. P¹, P², P⁵ or P⁶) may beindependently selected from optionally substituted —Si(C₁-C₆-alkyl)₃,optionally substituted —C(O)—C₁-C₆-alkyl, optionally substituted—C(O)-aryl, optionally substituted —C(O)—OC₁-C₆-alkyl, —C(O)—O-allyl,—C(O)—O—CH₂-fluorenyl, optionally substituted —C(aryl)₃, optionallysubstituted —(C₁-C₃-alkylene)-aryl, optionally substituted—C(O)OCH₂-aryl and —C₁-C₄-alkyl-O—C₁-C₄-alkyl. Where two hydroxyl groupsare attached to neighbouring carbon atoms (e.g. P⁵ and P⁶), they may bejointly protected with an optionally substituted —C(C₁-C₄-alkyl)₂-group.

A protecting group for an amino group (e.g. P³, P⁴, P⁷ or P⁸) may ateach occurrence be independently selected from —C(O)OC₁-C₆-alkyl,optionally substituted —C(O)OCH₂-aryl, —C(O)—O-allyl,—C(O)—O—CH₂-fluorenyl, optionally substituted —C(aryl)₃, optionallysubstituted —(C₁-C₃-alkylene)-aryl, optionally substituted—C(O)—C₁-C₆-alkyl, optionally substituted —C(O)-aryl,—S(O)₂—C₁-C₆-alkyl, optionally substituted —S(O)₂-aryl and optionallysubstituted —Si(C₁-C₆-alkyl)₃.

Many of the protected starting compounds of Formula IIIa, IIIb or IIIcare known in the art and/or can be prepared by known methods. Forexample starting compounds of Formula IIIa, IIIb and IIIc may besynthesized from the parent nucleoside by protecting the hydroxy and/oramino groups with suitable protecting groups. The protecting groups cantypically be added and removed using conventional protecting groupmethodology, for example, as described in “Protective Groups in OrganicChemistry,” edited by J W F McOmie (1973); “Protective Groups in OrganicSynthesis,” 2^(nd) edition, T W Greene (1991); and “Protecting Groups”,3^(rd) addition P. J Koscienski (1995).

It will typically be necessary to prepare the compounds of formulaeIIIa, IIIb and IIIc by first protecting the 5′-hydroxy group of theparent nucleoside with a protecting group which is orthogonal to thosewhich will be used to protect the 3′ and/or 2′-hydroxy and/or aminogroup (i.e. a group which can be removed from the 5′-hydroxyl groupwithout also removing the desired 3′-hydroxyl, 2′-hydroxyl and/or aminoprotecting groups). Simultaneously or subsequently, the 3′, 2′-hydroxyland/or amino groups are protected with the desired protecting group(s)and the 5′-hydroxyl protecting group can be removed to generate thecompound of formula IIIa, IIIb or IIIc. Certain protecting groups can besimultaneously introduced onto the 3′ and/or 2′-hydroxyl and 5′-hydroxyland optionally the amino groups and then selectively removed from the 5′hydroxyl group without being removed from the 3′ and/or 2′-hydroxyl andoptionally the amino groups.

According to some embodiments, P¹ is independently selected fromoptionally substituted —Si(C₁-C₆-alkyl)₃, optionally substituted—C(O)—C₁-C₆-alkyl, optionally substituted —C(O)-aryl, optionallysubstituted —C(O)—OC₁-C₆-alkyl, —C(O)—O-allyl, —C(O)—O—CH₂-fluorenyl,optionally substituted —C(aryl)₃, optionally substituted—(C₁-C₃-alkylene)-aryl, optionally substituted —C(O)OCH₂-aryl and—C₁-C₄-alkyl-O—C₁-C₄-alkyl.

P¹ may be independently selected from optionally substituted—Si(C₁-C₆-alkyl)₃, optionally substituted —C(O)—OC₁-C₆-alkyl andoptionally substituted —C(O)OCH₂-aryl, —C(O)—O-allyl. Preferably, P¹ isselected from —C(O)O-tBu, —C(O)O-benzyl and —C(O)OCH₂-allyl. Thus, P¹may be —C(O)OCH₂-aryl. P¹ may be —C(O)O-tBu.

Alternatively, P¹ may be independently selected from optionallysubstituted —C(O)—C₁-C₆-alkyl and optionally substituted —C(O)-aryl,e.g. P¹ may be independently selected from benzoyl and acetyl.

In a further alternative, P¹ may be optionally substituted—Si(C₁-C₆-alkyl)₃. P¹ may be —Si(C₁-C₄-alkyl)₃. The alkyl groups may beunsubstituted. P¹ may be t-butyldimethylsilyl.

According to some embodiments, P² is independently selected fromoptionally substituted —Si(C₁-C₆-alkyl)₃, optionally substituted—C(O)—C₁-C₆-alkyl, optionally substituted —C(O)-aryl, optionallysubstituted —C(O)—OC₁-C₆-alkyl, —C(O)—O-allyl, —C(O)—O—CH₂-fluorenyl,optionally substituted —C(aryl)₃, optionally substituted—(C₁-C₃-alkylene)-aryl, optionally substituted —C(O)OCH₂-aryl and—C1-C₄-alkyl-O—C₁-C₄-alkyl.

P² may be independently selected from optionally substituted—Si(C1-C₆-alkyl)₃, optionally substituted —C(O)—OC₁-C₆-alkyl andoptionally substituted —C(O)OCH₂-aryl, —C(O)—O-allyl. Preferably, P² isselected from —C(O)O-tBu, —C(O)O-benzyl and —C(O)OCH₂-allyl. Thus, P²may be —C(O)OCH₂-aryl. P² may be —C(O)O-tBu.

Alternatively, P² may be independently selected from optionallysubstituted —C(O)—C₁-C₆-alkyl and optionally substituted —C(O)-aryl,e.g. P² may be independently selected from benzoyl and acetyl.

In a further alternative, P² may be optionally substituted—Si(C₁-C₆-alkyl)₃. P² may be —Si(C₁-C₄-alkyl)₃. The alkyl groups may beunsubstituted. P² may be t-butyldimethylsilyl.

P³ may be independently selected from —C(O)OC₁-C₆-alkyl, optionallysubstituted —C(O)OCH₂-aryl, —C(O)—O-allyl, —C(O)—O—CH₂-fluorenyl,optionally substituted —C(aryl)₃, optionally substituted—(C₁-C₃-alkylene)-aryl, optionally substituted —C(O)—C₁-C₆-alkyl,optionally substituted —C(O)-aryl, —S(O)₂—C₁-C₆-alkyl, optionallysubstituted —S(O)₂-aryl and optionally substituted —Si(C₁-C₆-alkyl)₃.

P³ may be independently selected from —C(O)OC₁-C₆-alkyl, optionallysubstituted —C(O)OCH₂-aryl, —C(O)—O-allyl, optionally substituted—C(aryl)₃, and optionally substituted —Si(C₁-C₆-alkyl)₃. Preferably, P³is selected from —C(O)O-tBu, —C(O)O— benzyl and —C(O)OCH₂-allyl. Thus,P³ may be —C(O)OCH₂-aryl.

Alternatively, P³ may be independently selected from optionallysubstituted —C(O)—C₁-C₆-alkyl and optionally substituted —C(O)-aryl,e.g. P³ may be independently selected from benzoyl and acetyl.

In another alternative, P³ is H.

P⁴ may be independently selected from H, —C(O)OC₁-C₆-alkyl, optionallysubstituted —C(O)OCH₂-aryl, —C(O)—O-allyl, —C(O)—O—CH₂-fluorenyl,optionally substituted —C(aryl)₃, optionally substituted—(C₁-C₃-alkylene)-aryl, optionally substituted —C(O)—C₁-C₆-alkyl,optionally substituted —C(O)-aryl, —S(O)₂—C₁-C₆-alkyl, optionallysubstituted —S(O)₂-aryl and optionally substituted —Si(C₁-C₆-alkyl)₃.

Preferably, P⁴ is H.

It may be that P³ and P⁴ are each H. It may be that P³ and P⁴ are each Hand P² is —C(O)O-tBu. It may be that P³ and P⁴ are each H and P² ist-butyldimethylsilyl.

According to some embodiments, P⁵ and P⁶ are each independently selectedfrom optionally substituted —Si(C₁-C₆-alkyl)₃, optionally substituted—C(O)—C₁-C₆-alkyl, optionally substituted —C(O)-aryl, optionallysubstituted —C(O)—OC₁-C₆-alkyl, —C(O)—O— allyl, —C(O)—O—CH₂-fluorenyl,optionally substituted —C(aryl)₃, optionally substituted—(C₁-C₃-alkylene)-aryl, optionally substituted —C(O)OCH₂-aryl and—C₁-C₄-alkyl-O—C₁-C₄-alkyl; or P⁵ and P⁶ together form a optionallysubstituted —C(C₁-C₄-alkyl)₂-group. P⁵ and P⁶ may be the same.

P⁵ and P⁶ may each be selected from optionally substituted—Si(C₁-C₆-alkyl)₃, optionally substituted —C(O)—OC₁-C₆-alkyl andoptionally substituted —C(O)OCH₂-aryl, —C(O)—O-allyl. Preferably, P⁵ andP⁶ are each selected from —C(O)O-tBu, —C(O)O— benzyl and—C(O)OCH₂-allyl. Thus, P⁵ and P⁶ may each be —C(O)OCH₂-aryl. P⁵ and P⁶may each be —C(O)O-tBu.

Alternatively, P⁵ and P⁶ may each be selected from optionallysubstituted —C(O)—C₁-C₆-alkyl and optionally substituted —C(O)-aryl,e.g. P⁵ and P⁶ may each be selected from benzoyl and acetyl.

In a further alternative, P⁵ and P⁶ may each be optionally substituted—Si(C₁-C₆-alkyl)₃. P² may be —Si(C₁-C₄-alkyl)₃. The alkyl groups may beunsubstituted. P⁵ and P⁶ may each be t-butyldimethylsilyl.

Preferably, however, P⁵ and P⁶ together form a optionally substituted—C(C₁-C₄-alkyl)₂-group. It may be that P⁵ and P⁶ together form a—C(Me)₂-group.

P⁷ may be independently selected from —C(O)OC₁-C₆-alkyl, optionallysubstituted —C(O)OCH₂-aryl, —C(O)—O-allyl, —C(O)—O—CH₂-fluorenyl,optionally substituted —C(aryl)₃, optionally substituted—(C₁-C₃-alkylene)-aryl, optionally substituted —C(O)—C₁-C₆-alkyl,optionally substituted —C(O)-aryl, —S(O)₂—C₁-C₆-alkyl, optionallysubstituted —S(O)₂-aryl and optionally substituted —Si(C₁-C₆-alkyl)₃.

P⁷ may be independently selected from —C(O)OC₁-C₆-alkyl, optionallysubstituted —C(O)OCH₂-aryl, —C(O)—O-allyl, optionally substituted—C(aryl)₃, and optionally substituted —Si(C₁-C₆-alkyl)₃. Preferably, P⁷is selected from —C(O)O-tBu, —C(O)O— benzyl and —C(O)OCH₂-allyl. Thus,P⁷ may be —C(O)OCH₂-aryl.

Alternatively, P⁷ may be independently selected from optionallysubstituted —C(O)—C₁-C₆-alkyl and optionally substituted —C(O)-aryl,e.g. P⁷ may be independently selected from benzoyl and acetyl.

In another alternative, P⁷ is H.

Likewise, P⁸ may be independently selected from H, —C(O)OC₁-C₆-alkyl,optionally substituted —C(O)OCH₂-aryl, —C(O)—O-allyl,—C(O)—O—CH₂-fluorenyl, optionally substituted —C(aryl)₃, optionallysubstituted —(C₁-C₃-alkylene)-aryl, optionally substituted—C(O)—C₁-C₆-alkyl, optionally substituted —C(O)-aryl,—S(O)₂—C₁-C₆-alkyl, optionally substituted —S(O)₂-aryl and optionallysubstituted —Si(C₁-C₆-alkyl)₃.

Preferably, P⁸ is H.

It may be that P⁷ and P⁸ are each H. It may be that P⁷ and P⁸ are H andP⁵ and P⁶ together form a —C(Me)₂-group.

The group optionally substituted —Si(C₁-C₆-alkyl)₃ may be a—Si(C₁-C₄-alkyl)₃ group. The group is (i.e. the alkyl groups are)preferably unsubstituted. Illustrative examples include triethylsilyland t-butyl-dimethylsilyl.

The group optionally substituted —C(O)—C₁-C₆-alkyl may be a—C(O)—C₁-C₆-alkyl group. The group (i.e. the alkyl group) is preferablyunsubstituted. Illustrative examples include acetyl and propionyl.

The group optionally substituted —C(O)-aryl may be a —C(O)-phenyl group.The group (i.e. the phenyl group) is preferably unsubstituted.Illustrative examples include benzoyl.

The group optionally substituted —C(O)—OC₁-C₆-alkyl may be a—C(O)—OC₁-C₄-alkyl group. The group (i.e. the alkyl group) is preferablyunsubstituted. Illustrative examples include —C(O)—O-methyl and—C(O)—O-ethyl. A particularly preferred example is C(O)OtBu.

The group optionally substituted —(C₁-C₃-alkylene)-aryl is preferably anoptionally substituted benzyl group. Illustrative examples includebenzyl, phenethyl, 4-methoxy benzyl, 4-nitrobenzyl, 4-bromobenzyl,2,3-dimethoxybenzyl and 2,4-dimethoxybenzyl.

The group optionally substituted —C(O)OCH₂-aryl is preferably anoptionally substituted —C(O)Obenzyl group. Illustrative examples include—C(O)Obenzyl and —C(O)O-(4-methoxybenzyl).

The group optionally substituted —C₁-C₄-alkyl-O—C₁-C₄-alkyl may be a—C₁-C₂-alkyl-O—C₁-C₂-alkyl group. The group is (i.e. the alkyl groupsare) preferably unsubstituted. Illustrative examples includemethoxy-methyl (MOM) and 2-methoxy-ethoxy-methyl (MEM).

The group optionally substituted —S(O)₂—C₁-C₆-alkyl may be a—S(O)₂—C₁-C₄-alkyl group. The group (i.e. the alkyl group) is preferablyunsubstituted. Illustrative examples include methanesulfonate.

The group optionally substituted —S(O)₂-aryl may be a —S(O)₂-phenylgroup. Illustrative examples include phenylsulfonate,4-methylphenylsulfonate and 4-nitro phenylsulfonate.

The group optionally substituted —C(aryl)₃ may be a —C(phenyl)₃ group.Illustrative examples include trityl.

Where two or more of P², P³ and P⁴ or P⁵, P⁶, P⁷ and P⁸ are protectinggroups, the deprotection step may comprise two or three individualdeprotection reactions. This is the case where two or three differentprotecting groups are used and where those two or three protectinggroups cannot be removed under the same conditions.

It may be, however, that the deprotection step comprises a singledeprotection reaction in which all protecting groups are removed. Thus,it may be that P² and P³ are protecting groups which can be removedunder the same conditions. It may be that P² and P³ are the same.Likewise, it may be that P⁵ and P⁶ are protecting groups which can beremoved under the same conditions. It may be that P⁵ and P⁶ are thesame.

Throughout this specification, ‘diastereomerically enriched form’ and‘substantially diastereomerically pure form’ means a diastereoisomericpurity of greater than 95%. ‘Diastereomerically enriched form’ and‘substantially diastereomerically pure form’ may mean adiastereoisomeric purity of greater than 98%, greater than 99% orgreater than 99.5%.

Any of the aforementioned alkyl and aryl (e.g. phenyl, including thephenyl groups in benzyl groups) groups, are optionally substituted,where chemically possible, by 1 to 3 substituents which are eachindependently at each occurrence selected from the group consisting of:oxo, ═NR^(a), ═NOR^(a), halo, nitro, cyano, NR^(a)R^(a),NR^(a)S(O)₂R^(a), NR^(a)CONR^(a)R^(a), NR^(a)CO₂R^(a), OR^(a); SR^(a),SOR^(a), SO₃R^(a), SO₂R^(a), SO₂NR^(a)R^(a), CO₂R^(a) C(O)R^(a),CONR^(a)R^(a), C₁-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkenyl, and C₁-C₄haloalkyl; wherein R^(a) is independently at each occurrence selectedfrom H, C₁-C₄ alkyl and C₁-C₄ haloalkyl.

It may be that any of the aforementioned alkyl groups is unsubstituted.

It may be that any of the aforementioned aryl groups (e.g. phenyl,including the phenyl groups in benzyl groups) are optionallysubstituted, where chemically possible, by 1 to 3 substituents which areeach independently at each occurrence selected from the group consistingof: halo, nitro, cyano, NR^(a)R^(a), NR^(a)S(O)₂R^(a),NR^(a)CONR^(a)R^(a), NR^(a)CO₂R^(a), OR^(a); SR^(a), SOR^(a), SO₃R^(a),SO₂R^(a), SO₂NR^(a)R^(a), CO₂R^(a) C(O)R^(a), CONR^(a)R^(a),C₁-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkenyl, and C₁-C₄ haloalkyl; whereinR^(a) is independently at each occurrence selected from H, C₁-C₄ alkyland C₁-C₄ haloalkyl.

It may be that any of the aforementioned aryl (e.g. phenyl, includingthe phenyl groups in benzyl groups) groups are optionally substituted by1 to 3 substituents which are each independently at each occurrenceselected from the group consisting of: halo, nitro, OR^(a); C₁-C₄-alkyl,C₁-C₄ haloalkyl; wherein R^(a) is independently at each occurrenceselected from H, C₁-C₄ alkyl and C₁-C₄ haloalkyl.

Aryl groups have from 6 to 20 carbon atoms as appropriate to satisfyvalency requirements. Aryl groups are carbocyclic groups which satisfythe Huckel rule (i.e. they contain a carbocyclic ring system containing2(2n+1)π electrons). Aryl groups may be optionally substituted phenylgroups, optionally substituted biphenyl groups, optionally substitutednaphthalenyl groups or optionally substituted anthracenyl groups.Equally, aryl groups may include non-aromatic carbocyclic portions.Preferably an aryl group is an optionally substituted phenyl group.

Alkyl groups may be straight chain or branched. Thus, for example, a C₄alkyl group could be n-butyl, i-butyl or t-butyl.

Step a) of the first, second and third aspects may be conducted in anorganic solvent (S1). Organic solvents include but are not limited toethers (e.g. tetrahydrofuran, dioxane, diethyl ether,methyl-t-butylether); ketones (e.g. acetone and methyl isobutyl ketone);halogenated solvents (e.g. dichloromethane, chloroform and1,2-dichloroethane); and amides (e.g. DMF, NMP); or mixtures thereof.Where step a) is conducted in the presence of a Grignard reagent, theorganic solvent is preferably an ether. Most preferably, the solvent istetrahydrofuran.

Where step a) of the first aspect is conducted in the present of anitrogen base, the organic solvent is most preferably a halogenatedsolvent or an amide.

The reaction is typically conducted at a suitable temperature, e.g fromabout −5° C. to about 40° C. Preferably, the reaction temperature isabout 25° C. to about 30° C. The reaction may be allowed to stir for aperiod of time from about 15 mins to about 16 h and preferably fromabout 30 mins to about 60 mins.

The processes of the invention may also involve deprotection of thehydroxy and amino protecting groups.

It may be that the deprotection step (step b) is carried out withoutpurifying the product of step a).

Where a protecting group is acid sensitive (e.g. trityl, C(O)OtBu, MOM,MEM, 2,4-dimethoxybenzyl, 2,3-dimethoxybenzyl, —C(Me)₂-) thedeprotection step can be conducted using a suitable acid. The acid maybe a Bronsted acid (e.g. TFA, phosphoric acid, HCl, or formic acid) or aLewis acid (e.g. ZnBr₂, CeCl₃). Lewis acids (e.g. ZnBr₂) are lesspreferred. HCl is likewise less preferred. Preferably, the acid is TFA.

Where a protecting group is base sensitive, e.g. acetyl, benzoyl, thedeprotection step can be conducted using a suitable base, e.g. aqueousNH₃ or aqueous NaOH. Base sensitive groups may be less preferred.

Where a protecting group is a silyl group (e.g. triethylsilyl ort-butyldimethylsilyl, the deprotection step can be conducted using asuitable acid (e.g. TFA) or using a suitable fluorine source (e.g.tetrabutylammonium fluoride, fluorosilicic acid, HF).

Where a protecting group is a benzyl group or a C(O)Obenzyl group, thedeprotection step can be conducted using H₂ and a suitable catalyst(e.g. Pd/C). Such protecting groups may be less preferred.

Where a protecting group is a 4-methoxy-benzyl, 2,3-dimethoxybenzyl,2,4-dimethoxybenzyl or C(O)O-(4-methoxybenzyl) the deprotection step canbe performed using a suitable oxidizing agent (e.g.meta-chloroperbenzoic acid).

Where a protecting group is —C(O)—O-allyl, the deprotection step can beperformed using (PPh₃)₄Pd.

Where a protecting group is —C(O)—O—CH₂-fluorenyl, the deprotection stepcan be performed using piperidine.

The deprotection step may be conducted in an organic solvent or amixture thereof. Exemplary organic solvents include, but are not limitedto halogenated solvents (e.g. dichloromethane, chloroform,dichloroethane); alcohols (e.g. methanol, ethanol, isopropanol) andethers (e.g. tetrahydrofuran, diethyl ether).

Where the deprotection step is carried out in the presence of an acid(e.g. TFA, the organic solvent is preferably a halogenated solvent, e.g.dichloromethane.

The deprotection reaction may be carried out at a temperature in therange of, for example −10° C. to about 30° C., e.g. to about 10° C. Aconvenient temperature to carry out the reaction is −5° C. to 5° C. Thereaction may be allowed to stir for a period of time from about 15 minsto about 16 hours and preferably from about 1 hour to about 4 hours, andmore preferably from about 2 hours to about 3 hours.

Where step b) is achieved using a C₁-C₄-alcohol and/or water (e.g. amixture of isopropyl alcohol (IPA) and water), the reaction mixture maybe heated, e.g. to a temperature from 30° C. to 90° C. or to atemperature from 60° C. to 85° C.

Where, the deprotection is performed in the presence of an acid (e.g.TFA), isolation of the product obtained after the deprotection istypically done by quenching the excess acid used in deprotection stepand extracting the product with a water immiscible organic solvent andrecovering the product by evaporation of the organic solvent.

Examples of water immiscible organic solvents useful in extractioninclude esters such as ethyl acetate, methyl acetate, isopropyl acetateand the like; chlorinated solvents such as dichloromethane, chloroformand the like; aromatic hydrocarbon solvents such as toluene, xylene andthe like; preferably ethyl acetate.

In certain embodiments, it may still be desirable to purify the ProTideobtained from the process of the first aspect of the invention.Likewise, it may still be desirable to purify the compound of formulaIIa obtained from the process of the fourth aspect of the invention.Methods of purification are well known to those skilled in the art andinclude chromatography (e.g. column chromatography), recrystallisationand distillation. In other embodiments, no purification is necessary.

It will be understood that various modifications may be made to theembodiments disclosed herein. Therefore the above description should notbe construed as limiting, but merely as exemplifications of preferredembodiments. For example, the functions described above and implementedas the best mode for operating the present invention are forillustration purposes only. Other arrangements and methods may beimplemented by those skilled in the art without departing from the scopeand spirit of this invention. Moreover, those skilled in the art willenvision other modifications within the scope and spirit of thespecification appended hereto.

The following abbreviations are used throughout this specification:

ACN—acetonitrile AIBBr—acetoxy isobutyryl bromide BOC—t-butylcarbonateDCM—dichloromethane DMAP—N,N-dimethyl-4- DMF—N,N-dimethylformamideaminopyridine eq.—molar equivalents FUDR—5-fluoro-2′-deoxyuridineIPA—isopropyl alcohol MEM—2-methoxyethoxymethyl MOM—methoxymethylMTBE—methyl-t-butylether NMP—N-methyl-2-pyrrolidone Np—1-naphthylPTSA—para-toluene sulfonic RT—room temperature (tosic) acidTBAF—tetrabutylammonium TBDMS—tert-butyldimethylsilyl fluorideTEA—triethylamine Tf—trifluoromethylsulfonate (triflate)TFA—trifluoroacetic acid THF—tetrahydrofuran

V is used to denote volume (in mL) per weight (in g) starting material.So if there was 1 g of starting material, 10 V would mean 10 mL of theindicated liquid.

EXAMPLES

The present invention is further illustrated by the following examples,which are provided by way of illustration only and should not beconstrued to limit the scope of the invention.

Example 1: Preparation of Diastereoisomeric Mixture of2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphoryl amino] propionicacid benzyl ester 5 (An Illustrative Example of a Compound of FormulaIIb)

To a stirred mixture of L-alanine benzyl ester hydrochloride 1 (100 g)in methylene chloride (1 L) was added phenyl dichlorophosphate 2 (77 mL)at 25-35° C. and the resulting mixture was cooled to −70° C. to −78° C.,triethylamine (130.5 mL) was added and the mixture was stirred for 1hour at same temperature. Reaction mass temperature was raised to 25-35°C. and allowed to stir for 2 hours. After reaction completion,concentrated the reaction mass under vacuum at below 35° C. to obtainresidue. To the obtained residue was added diisopropyl ether (2 L) at25-35° C. and stirred for 30 min at same temperature. Filtered thereaction mass and washed with diisopropyl ether (500 mL) followed byconcentrating the filtrate under vacuum at below 35° C. to obtainphenyl-(benzoxy-L-alaninyl)-phosphorochloridate 3. The obtained compoundwas dissolved in methylene chloride (1 L) at 25-35° C. and cooled to −5°C. to −10° C. To the reaction mass pentafluorophenol 4 (85.5 g),triethylamine (65.2 mL) were added at same temperature and stirred for 2hrs. After reaction completion, concentrated the reaction mass undervacuum at below 35° C. and charged ethyl acetate (1 L) at 25-35° C. andstirred for 30 min at same temperature. Filtered the solids and washedwith ethyl acetate (1 L). To the filtrate was given water (1 L), 10%sodium carbonate (2×1 L), brine (1 L) washings and dried the organiclayer with anhydrous sodium sulphate, concentrated under vacuum at35-45° C. to obtain diastereoisomeric mixture of title compound 5 as awhite colored semi solid.

Yield: 210 g

Chiral Purity by HPLC (% area): 33.74:66.26% (R_(P):S_(P))

Example 2: Separation of S_(p)-Diastereoisomer of2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphoryl amino] propionicacid benzyl ester 5 (An Illustrative Example of a Compound of FormulaIIb)

To a diastereoisomeric mixture of compound 5 (210 g;R_(P):S_(P)-33.74:66.26%) was charged 20% ethyl acetate in hexane (1.2L) at 25-35° C. and stirred for 1 hrs. Filtered the solids and washedwith 20% ethyl acetate in hexane (300 mL) to obtain a mixture ofdiastereoisomeric mixture of compound 5.

Yield: 112 g

Chiral Purity by HPLC (% area): 22.13:77.87% (R_(P):S_(P))

Filtrate was concentrated under vacuum to obtain a diastereoisomericmixture of compound of 5 (75 g; R_(P): S_(P)—65.43:34.57%).

To a diastereoisomeric mixture of the compound of formula IIb (112 g;R_(P):S_(P)—22.13:77.87%) was charged 20% ethyl acetate in hexane (1.2lit) at 25-35° C. and stirred for 1 hrs. Filtered the solids and washedwith 20% ethyl acetate in hexane (300 ml) to obtain substantially pureS_(p)-diastereoisomer of compound 5.

Yield: 80 g

Chiral Purity by HPLC (% area): 0.20:99.80% (R_(P):S_(P))

¹H NMR (300 MHz, DMSO-d₆): 7.18-7.41 (m, 10H), 6.91-6.99 (d, 1H), 5.10(s, 2H), 4.01-4.11 (m, 1H), 1.30-1.32 (d, 3H)

ESI-MS (m/z): 524 (M+1)

Filtrate was concentrated under vacuum to obtain a diastereoisomericmixture of compound 5 (28 g; R_(P):S_(P)—80.77:19.23%).

Example 3: Enrichment of2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphoryl amino] propionicacid benzyl ester 5 S-isomer (An Illustrative Example of a Compound ofFormula IIb)

To a stirred solution of2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphoryl amino] propionicacid benzyl ester 5 (75 g; R_(P):S_(P)—65.43:34.57%) in 20% ethylacetate in hexane (1.1 L), triethyl amine (7.5 mL) was added at 25-35°C. and stirred for 6 hrs at same temperature. After reaction completion,reaction mass was quenched in to a water (750 mL) and extracted withethyl acetate (750 mL). Organic layer was dried with anhydrous sodiumsulphate and concentrated under vacuum to afford title compound as asolid.

Yield: 45 g

Chiral Purity by HPLC (% area): 91.29:8.71% (S_(P):R_(P))

To the above obtained R_(p) and S_(p)-diastereoisomeric mixture of2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphoryl amino] propionicacid benzyl ester 5 (45 g; R_(P):S_(P)—8.71:91.29%) was slurred in 20%ethyl acetate in hexane (1.1 L) at 25-30° C. and stirred for 1 hr atsame temperature. Filtered the solid and washed with 20% ethyl acetatein hexane (225 ml) to obtain S_(p)-diastereoisomer of the title compoundas a solid.

Yield: 19 g

Chiral Purity by HPLC (% area): 99.92:0.08% (S_(P):R_(p))

Example 4: Preparation of Diastereoisomeric Mixture of2-[(4-nitrophenoxy)-phenoxy-phosphorylamino] propionic acid benzyl ester7 (An Illustrative Example of a Compound of Formula IIb

To a stirred mixture of L-alanine benzyl ester hydrochloride 1 (50 g) inmethylene chloride (500 mL) was added phenyl dichlorophosphate 2 (54 g)at 25-35° C. and the resulting mixture was cooled to −70° C. to −78° C.,added triethyl amine (65.2 mL) and stirred for 1 hour at sametemperature. Reaction mass temperature was raised to 25-35° C. andallowed to stir for 2 hrs. After reaction completion, concentrated thereaction mass under vacuum at below 35° C. to obtain residue. To theobtained residue was added diisopropyl ether (1 L) at 25-35° C. andstirred for 30 min at same temperature. Filtered the reaction mass andwashed with diisopropyl ether (250 mL) followed by concentrating thefiltrate under vacuum at below 35° C. to obtainphenyl-(benzoxy-L-alaninyl)-phosphorochloridate 3. The obtained compoundwas dissolved in methylene chloride (500 mL) at 25-35° C. and cooled to−5° C. to −10° C. To the reaction mass 4-nitrophenol 6 (27.5 g),triethyl amine (65.2 mL) was added at same temperature and stirred for 2hrs. After reaction completion, concentrated the reaction mass undervacuum at below 35° C. and charged ethyl acetate (500 mL) at 25-35° C.and stirred for 30 min at same temperature. Filtered the solids andwashed with ethyl acetate (500 mL). To the filtrate was given water (500mL), 10% sodium carbonate (2×500 mL), brine (500 mL) washings and driedthe organic layer with anhydrous sodium sulphate, concentrated undervacuum at 35-40° C. to obtain diastereoisomeric mixture of titlecompound 7 as a thick oily liquid.

Yield: 90 g

Chiral Purity by HPLC (% area): 45.6:54.94% (R_(P):S_(P))

The above obtained diastereoisomeric mixture of2-[(4-nitrophenoxy)-phenoxy-phosphorylamino] propionic acid benzyl ester7 (40 g; R_(p):S_(p)-45.6:54.94%) was separated in to pure S_(p) andR_(p) diastereoisomers by preparative HPLC and concentrated the purefractions under vacuum to obtain S_(p) and R_(p) diastereoisomersseparately.

Yield: S_(p)-diastereoisomer: 8 g,

¹H NMR (300 MHz, CDCl₃): 8.15-8.19 (d, 2H), 7.15-7.37 (m, 12H), 5.12 (s,2H), 4.02-4.24 (m, 2H), 1.39-1.42 (d, 3H)

ESI-MS (m/z): 479 (M+Na)

R_(p)-diastereoisomer: 6 g,

¹H NMR (300 MHz, CDCl₃): 8.08-8.13 (d, 2H), 7.15-7.34 (m, 12H), 5.10 (s,2H), 4.48-4.56 (m, 1H), 4.11-4.20 (m, 1H), 1.39-1.41 (d, 3H)

ESI-MS (m/z): 457 (M+1)⁺

S_(p) and R_(p)-diastereoisomers mixture: 20 g

Example 5—Preparation of(Sp)-2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphoryl amino]propionic acid benzyl ester 5 (An Illustrative Example of a Compound ofFormula IIb)

To a stirred mixture of L-Alanine Benzyl ester. HCl 1 (100 g) in 1000 mLof methylene dichloride was added phenyl dichlorophosphate 2 (97.8 g)into reaction mass at 30° C. The mixture was cooled to −20° C. andtriethylamine (93.8 g) was added slowly, maintaining the temperature at−20° C. The reaction was stirred for h at −20° C., then warmed to 10° C.(10±5) and stirred for a further 1.5 h.

A solution of pentafluorophenol 4 (85.3 g) in 100 mL of methylenedichloride was slowly added at 10° C. followed by trimethylamine (46.8g) which is added slowly, maintaining the temperature at 10° C. Slowlyadd 46.9 g of triethylamine into reaction mass at 10° C. (10±5) undernitrogen atmosphere. The mixture was stirred for 2 h at 10° C. beforebeing quenched by slow addition of 0.5 N HCl solution, maintaining thetemperature at 10° C. After warming to room temperature the mixture wasseparated and the organics was washed with a saturated bicarbonatesolution, distilled water and brine before being concentrated in vacuo.

The crude mixture was suspended in 1500 mL of 20% ethyl acetate inn-heptane at 25° C. Triethylamine (12.2 g) was added and the mixture wasstirred at 25° C. The mixture was filtered and the solid dissolved in2500 mL ethyl acetate which was washed with water and brine andconcentrated in vacuo. The solid was suspended in 1200 mL of 20% ethylacetate in n-heptane, stirred for 45-60 min and filtered. The materialwas dried under vacuum to provide the desired product 5-(Sp). Yields arein the range 40 to 80% and the diastereoisomeric purity is over 99%.

Example 6: Preparation of Diastereoisomeric Mixture of2-[(2,3,4,5,6-pentafluorophenoxy)-naphth-1-oxy-phosphoryl amino]propionic acid benzyl ester 12 (An Illustrative Example of a Compound ofFormula IIa)

Alpha-naphthol 8 (100 g) was dissolved in DCM (1 L) at 25° C. and POCl₃9 (1.1 eq) was added at 25° C. and stirred for 10 min before the mixturewas cooled to −70° C. and stirred for 10 min. Triethylamine (1.1 eq.)was added slowly maintaining the temperature at below −70° C. and themixture was stirred for 1 h at −70° C. The mixture was warmed to 25° C.and stirred for 1 h before being cooled to −50° C. L-alanine benzylester 1 (HCl salt; 1 eq.) was added to the mixture which stirred for 10min before triethylamine (2.2 eq) in DCM (200 mL) was added at −50° C.over 30 minutes. The mixture was stirred for 1 h at −50° C. before beingwarmed to 25° C. and stirred for a further 1 h. The mixture was cooledto −10° C. and stirred for 10 min before pentafluorophenol 4 in DCM (200mL) was added to the reaction mass slowly at below −10° C. The mixturewas stirred at −10° C. for 10 min before triethylamine (1.1 eq.) wasadded over 30 min at −10° C. The mixture was stirred at −10° C. for 1 hbefore the mixture was warmed to 0° C. Water (1 L) was added and themixture was stirred for 30 min at 0° C. The mixture was warmed to 25° C.and stirred for 5-10 min before the organic layer was separated. Theaqueous layer was extracted with DCM (500 mL). The combined organiclayers were washed with 7% sodium bicarbonate solution (2×1 L) and theorganic layer was dried over anhydrous sodium sulphate before beingconcentrated in vacuo.

50% IPA/water (2.4 L) was added to the crude compound and stirred for 1h at 25° C. The solid compound was filtered and the wet cake was washedwith 50% IPA/water (500 mL) before being dried in vacuo. Again 50%IPA/water (2.4 L) was added to the crude compound and stirred for 1 h at25° C. before being filtered and the wet cake was again washed with 50%IPA/water (500 mL) before being dried in vacuo. The semi-dried compoundwas washed with cyclohexane (10 v/w) at 25-30° C. for 1 h before thesolid compound was washed with cyclohexane (2 L) and the wet compound 12was dried under vacuum at 55-60° C. ° C. for 12 h

Results:

-   -   Weight of the compound: 252 g    -   Overall yield: 66%    -   HPLC purity: 98.31% (diastereoisomeric ratio is 1:1)

³¹P NMR (202 MHz, CDCl₃): δ_(P) −1.35, −1.41; ¹H NMR (500 MHz, CDCl₃):δ_(H) 8.13-8.10 (1H, m, H—Ar), 7.90-7.88 (1H, m, H—Ar), 7.73 (1H,apparent d, J=8.5 Hz, H—Ar), 7.62-7.55 (3H, m, H—Ar), 7.45-7.41 (1H, m,H—Ar), 7.36-7.28 (5H, m, H—Ar), 5.01 (1H, apparent s, CH₂Ph), 5.12 (1H,q, J=12.5 Hz, CH₂Ph), 4.38-4.31 (1H, m, NHCHCH₃), 4.17-4.08 (1H, m,NHCHCH₃), 1.49, 1.47 (3H, 2×d, J=3.5 Hz, NHCHCH₃); MS (ES+) m/z: 574(M+Na⁺, 100%), Accurate mass: C₂₆H₁₉F₅NO₅P required 551.40 found 574.05(M+Na⁺); Reverse-phase HPLC, eluting with H₂O/MeOH in 20/80 in 35 min,F=1 mL/min, λ=254, two peaks for two diastereoisomers with t_(R)=12.96,14.48 min.

The diastereoisomers of compound 12 were separated by HPLC with BiotageIsolera using C18 SNAP Ultra (30 g) cartridge with a mixture of MeOH/H₂O(70%/30%) as an eluent to give: the fast eluting isomer (believed to bethe Rp diastereoisomer) and the slow eluting isomer (believed to be theSp diastereoisomer)

Note: Isomers are named as fast eluting (FE) and slow eluting (SE) basedon retention time on C18 (reversed phase) cartridge and HPLC analyticalcolumn.

Fast eluting isomer (believed to be the Rp diastereoisomer): ³¹P NMR(202 MHz, CDCl₃): δ_(P) −1.41; ¹H NMR (500 MHz, CDCl₃): δ_(H) 8.02 (1H,dd, J=7.0, 2.0 Hz, H—Ar), 7.79 (1H, dd, J=6.5, 3.0 Hz, H—Ar), 7.64 (1H,d, J=8.5 Hz, H—Ar), 7.53-7.45 (3H, m, H—Ar), 7.33 (1H, t, J=8.0 Hz,H—Ar), 7.28-7.23 (5H, m, H—Ar), 5.09 (s, 2H, CH₂Ph), 4.29-4.21 (1H, m,NHCHCH₃), 4.02-3.97 (1H, m, NHCHCH₃), 1.38 (3H, d, J=7.0 Hz, NHCHCH₃);MS (ES+) m/z: MS (ES+) m/z: 574 (M+Na⁺, 100%), Accurate mass:C₂₆H₁₉F₅NO₅P required 551.40 found 574.05 (M+Na⁺); Reverse-phase HPLC,eluting with H₂O/MeOH in 20/80 in 35 min, F=1 mL/min, λ=254,t_(R)=12.96.

Slow eluting isomer (believed to be the Sp diastereoisomer): ³¹P NMR(202 MHz, CDCl₃): δ_(P) −1.36; ¹H NMR (500 MHz, CDCl₃): δ_(H) 8.14-8.11(1H, m, H—Ar), 7.90-7.87 (1H, m, H—Ar), 7.74 (1H, d, J=8.0 Hz, H—Ar),7.60 (1H, d, J=8.0 Hz, H—Ar), 7.58-7.55 (2H, m, H—Ar), 7.44 (1H, t,J=8.0 Hz, H—Ar), 7.34-7.30 (5H, m, H—Ar), 5.12 (2H, q, J=12.5 Hz,CH₂Ph), 4.35-4.29 (1H, m, NHCHCH₃), 4.04-4.00 (1H, m, NHCHCH₃), 1.48(3H, d, J=7.0 Hz, NHCHCH₃); MS (ES+) m/z: MS (ES+) m/z: 574 (M+Na⁺,100%), Accurate mass: C₂₆H₁₉F₅NO₅P required 551.40 found 574.05 (M+Na⁺);Reverse-phase HPLC, eluting with H₂O/MeOH in 20/80 in 35 min, F=1mL/min, λ=254, t_(R)=14.48.

Example 7: Enrichment of S_(p)-Diastereoisomer of2-[(2,3,4,5,6-pentafluorophenoxy)-naphth-1-oxy-phosphoryl amino]propionic acid benzyl ester 12 Sp isomer (An Illustrative Example of aCompound of Formula IIb)

A 1:1 diastereoisomeric mixture of compound 12 (25 g) was dissolved in10% MTBE/n-Hexane (500 mL) and triethylamine (2.5 mL) was added to thereaction mass at 25° C. The mixture was stirred for 80 h at 30° C. Themixture was filtered and the wet cake was washed with 10% MTBE/n-hexane(75 mL) before being dried in vacuo 30 min. 50% IPA/water (200 mL) wasadded to above crude compound and stirred for 1 h at 25-35° C. beforebeing filtered. The wet cake was washed with 50% IPA/water (100 mL)before being dried in vacuo at 55-60° C. ° C. for 12 h

Result:

-   -   Wt. of the compound: 17 g    -   Yield: 68%    -   HPLC purity: 97.66%

Slow Eluting Isomer (Believed to be Sp-Diastereoisomer):

³¹P NMR (202 MHz, CDCl₃): δ_(P) −1.36; ¹H NMR (500 MHz, CDCl₃): δ_(H)8.14-8.11 (1H, m, H—Ar), 7.90-7.87 (1H, m, H—Ar), 7.74 (1H, d, J=8.0 Hz,H—Ar), 7.60 (1H, d, J=8.0 Hz, H—Ar), 7.58-7.55 (2H, m, H—Ar), 7.44 (1H,t, J=8.0 Hz, H—Ar), 7.34-7.30 (5H, m, H—Ar), 5.12 (2H, q, J=12.5 Hz,CH₂Ph), 4.35-4.29 (1H, m, NHCHCH₃), 4.04-4.00 (1H, m, NHCHCH₃), 1.48(3H, d, J=7.0 Hz, NHCHCH₃); MS (ES+) m/z: MS (ES+) m/z: 574 (M+Na⁺,100%), Accurate mass: C₂₆H₁₉F₅NO₅P required 551.40 found 574.05 (M+Na⁺);Reverse-phase HPLC, eluting with H₂O/MeOH in 20/80 in 35 min, F=1mL/min, λ=254, t_(R)=14.48.

The stereochemistry (Rp vs Sp) of the two compound 12 isomers describedabove has been assigned tentatively on the basis of comparison of ³¹Pchemical shift, 1H NMR spectra, and HPLC retention times of the NUC-3373isomers made using the compound 12 isomers with those of other ProTidesknown in the literature. As mentioned above, the stereochemistry ofphosphate stereocentre is inverted during the process of the inventionso the (Sp)-diastereoisomer of the compound of formula 12 will form the(Sp)-diastereoisomer of NUC-3373 and likewise the (R)-diastereoisomer ofthe compound of formula 12 will form the (R)-diastereoisomer ofNUC-3373. The stereochemical assignment is supported by powder X-raydiffraction and differential scanning calorimetry that has been carriedout on the two compound 12 isomers, but this is not in itselfdefinitive.

Example 8—Formation of Sp and Rp Isomers of NUC-3373

3′-BOC protected FUDR 16 can be made according to the following scheme.

Compound 16 can then be coupled with a compound of formula IIa.

Compound 16 (1 g) and the Sp isomer of compound 12 (1.2 eq) weredissolved in THF (10 mL) and the mixture was cooled to 0° C. t-Butylmagnesium chloride (2.5 eq, 2.0 M in THF) was added to the mixture over15 min. The mixture was warmed and stirred at 25° C. for 4 h. Themixture was cooled to 10° C. and sat. ammonium chloride solution (10 mL)was added. Ethyl acetate (10 mL) was added to the mixture and theorganic layer was separated. The aqueous layer was extracted with ethylacetate (5 mL). The combined organic layers were washed with deionisedwater (5 mL) followed by 20% sodium chloride solution (5 mL). Theorganic layers were dried over anhydrous sodium sulphate before beingconcentrated in vacuo to provide 2.16 g of compound 17 (100% crudeyield).

Crude compound 17 (1 g) was dissolved in DCM (5 mL) and cooled to 10° C.TFA (2 mL) was added slowly to the mixture, maintaining the temperatureat below 20° C. The mixture was warmed to 30° C. and the stirred for 6h. The mixture was cooled to 10° C. and deionized water (5 mL) was addedslowly, maintaining the temperature at below 20° C. After stirring for10 min the organic layer was separated and the aqueous layer wasextracted with DCM (5 mL). The combined organic layers were washed withdeionised water (2×5 mL), 7% sodium bicarbonate solution (2×5 mL) and20% sodium chloride solution (5 mL) before being dried with anhydroussodium sulphate (1 w/w) and concentrated in vacuo. Crude compound waspurified with column chromatography in ethyl acetate/DCM using silicagel (100-200 mesh). Pure compound was eluted in 50% Ethyl acetate/DCM to100% ethyl acetate. The combined pure fractions were concentrated invacuo before the compound slurry was washed with cyclohexane (5 mL).

Results:

-   -   Weight of NUC-3373 (Sp isomer): 9.3 g    -   Overall yield: 70%    -   HPLC purity: 96.86%

¹H-NMR (500 MHz, MeOD): δ_(H) 8.16-8.14 (m, 1H, H—Ar), 7.90-7.80 (m, 1H,H—Ar), 7.72-7.70 (m, 2H, H—Ar), 7.54-7.49 (m, 3H, H—Ar, H-6), 7.43(apparent t, 1H, J=8.0 Hz, H—Ar), 7.35-7.27 (m, 5H, H—Ar), 6.16-6.13 (m,1H, H-1′), 5.11 (AB system, J=12.0 Hz, 2H, OCH₂Ph), 4.35-4.33 (m, 2H,2×H-5′), 4.30-4.28 (m, 1H, H-3′), 4.14-4.08 (m, H, CHCH₃), 4.07-4.04 (m,1H, H-4′), 2.14-2.09 (m, 1H, H-2′), 1.74-1.68 (m, 1H, H-2′), 1.35 (d,J=7.0 Hz, 3H, CHCH₃);

¹³C-NMR (125 MHz, MeOD): δ_(C) 174.92 (d, ³J_(C-P)=3.75 Hz, C═O, ester),159.37 (d, ²J_(C-F)=25.9 Hz, C═O, base), 150.54 (d, ⁴J_(C-F)=4.0 Hz,C═O, base), 147.99 (d, ²J_(C-P)=7.1 Hz, C—Ar, Naph), 141.75 (d,¹J_(C-F)=232.1 Hz, CF-base), 137.18, 136.29 (C—Ar), 129.59, 129.36,128.90, 127.91 (CH—Ar), 127.83 (d, ³J_(C-P)=5.4 Hz, C—Ar, Naph), 127.59,126.52, 126.50, 126.18 (CH—Ar), 125.54 (d, ²J_(C-F)=34.1 Hz, CH-base),122.64 (CH—Ar), 116.29 (d, ³J_(C-P)=2.75 Hz, CH—Ar, Naph), 86.95 (C-1′),86.67 (d, ³J_(C-P)=8.1 Hz, C-4′), 72.12 (C-3′), 68.05 (OCH₂Ph), 67.85(d, ²J_(C-P)=5.3 Hz, C-5′), 51.96 (CHCH₃), 40.84 (C-2′), 20.52 (d,³J_(C-P)=7.5 Hz, CHCH₃).

³¹P-NMR (202 MHz, MeOD): δ_(P) 4.62;

¹⁹F NMR (470 MHz, MeOD): δ_(F)-167.19;

(ES+) m/z: Found: (M+Na⁺) 636.1520. C₂₉H₂₉N₃O₉FNaP required: (M⁺),613.15.

Reverse HPLC (Varian Pursuit XRs 5 C18, 150×4.6 mm) eluting with(H₂O/AcCN from 90/10 to 0/100) in 35 min., t_(R) 16.61 min.

The Rp isomer of NUC-3373 can be accessed by performing the aboveprocess but starting with the Rp diastereomer of compound 12:

¹H-NMR (500 MHz, MeOD): δ_(H) 8.17-8.15 (m, 1H, H—Ar), 7.91-7.88 (m, 1H,H—Ar), 7.72-7.69 (m, 2H, H—Ar), 7.56-7.52 (m, 2H, H—Ar, H-6), 7.50-7.48(m, 1H, H—Ar), 7.39 (apparent t, J=8.0 Hz, 1H, H—Ar), 7.35-7.28 (m, 5H,H—Ar), 6.16-6.09 (m, 1H, H-1′), 5.13 (s, 2H, OCH₂Ph), 4.35-4.25 (m, 3H,2×H-5′, H-3′), 4.14-4.08 (m, 1H, CHCH₃), 4.05-4.03 (m, 1H, H-4′),2.15-2.10 (m, 1H, H-2′), 1.74-1.68 (m, 1H, H-2′), 1.36 (d, J=7.0 Hz, 3H,CHCH₃);

¹³C-NMR (125 MHz, MeOD): δ_(C) 174.58 (d, ³J_(C-P)=5.0 Hz, C═O, ester),159.38 (d, ²J_(C-F)=26.3 Hz, C═O), 150.48 (C═O base), 147.80 (d,²J_(C-P)=6.5 Hz, C—Ar, Naph), 141.67 (d, ¹J_(C-F)=232.5 Hz, CF-base),137.15, 136.26 (C—Ar), 129.62, 129.40, 129.36, 128.96, 127.89 (CH—Ar),127.84 (d, ³J_(C-P)=5.5 Hz, C—Ar, Naph), 127.59, 126.57, 126.55, 126.21(CH—Ar), 125.61 (d, ²J_(C-F)=34.0 Hz, CH-base), 122.62 (CH—Ar), 116.55(d, ³J_(C-P)=3.75 Hz, CH—Ar, Naph), 86.97 (C-1′), 86.66 (d, ³J_(C-P)=7.5Hz, C-4′), 72.01 (C-3′), 68.07 (OCH₂Ph), 67.84 (d, ²J_(C-P)=5.0 Hz,C-5′), 51.83 (CHCH₃), 40.89 (C-2′), 20.42 (d, ³J_(C-P)=7.5 Hz, CHCH₃).

³¹P-NMR (202 MHz, MeOD): δ_(P) 4.27;

¹⁹F NMR (470 MHz, MeOD): δ_(F)-167.27;

(ES+) m/z: Found: (M+Na⁺) 636.1520. C₂₉H₂₉N₃O₉FNaP required: (M⁺),613.15.

Reverse HPLC (Varian Pursuit XRs 5 C18, 150×4.6 mm) eluting with(H₂O/MeOH from 90/10 to 0/100) in 35 min., t_(R) 16.03 min.

The stereochemistry (Rp vs Sp) of the two NUC-3373 isomers describedabove has been assigned tentatively on the basis of comparison of ³¹Pchemical shift, ¹H NMR spectra, and HPLC retention times with those ofother ProTides known in the literature. The stereochemistry of compound12 has been tentatively assigned based on which isomer of NUC-3373 thatisomer of compound 12 forms.

Example 9—Formation of Sp and Rp Isomers of NUC-7738

2′-TBDMS protected 3′-deoxyadenosine 21 can be made according to thefollowing scheme.

Compound 21 can then be coupled with a compound of formula IIb using thecoupling process conditions described in Example 8. To form NUC-7738,the TBDMS group can be removed using TFA in THF.

Adenosine (18) to Epoxide 19

One equivalent adenosine (18) was dissolved in 10 V acetonitrile and themixture was cooled to 15° C. 3.0 molar equivalents acetoxy isobutyrylbromide was added slowly at 15° C. The mixture was warmed to roomtemperature and stirred for 8 hours. The reaction was quenched withsodium bicarbonate solution and extracted with ethyl acetate. Thecombined organic layers were washed with 5% sodium chloride solution andthe organic layers were concentrated in vacuo.

The product was dissolved in 15 V methanol and 1 weight equivalent ofpotassium carbonate was added before stirring for 2 hours. The mixturewas concentrated in vacuo and the product was washed with water beforedrying the product under vacuum at 60° C. to provide 2′,3′-anhydroadenosine in a yield of 70-85%.

1 equivalent of 2′,3′-anhydro adenosine and 1.6 equivalents imidazolewere dissolved in 5V DMF. The mixture was cooled to 15° C. and 0.8equivalents TBDMSCI was added. The mixture was stirred for 1 to 2 hoursat 30° C. before a further 0.4 equivalents imidazole and 0.4 equivalentsTBDMSCI were added. The mixture was stirred for a further 1 to 2 hoursat 30° C. before water was added (5V). The mixture was extracted withethyl acetate. The combined organic layers were sequentially washed with7% sodium bicarbonate solution, water and 5% sodium chloride solutionbefore being concentrated in vacuo. The product was washed with heptanebefore being dried under vacuum at 50° C. to obtain epoxide 19 in 75-90%yield.

Epoxide 19 to 5′-Silyl Cordycepin 21

1 Equivalent of epoxide 19 was dissolved in a mixture of DMSO (5V) andTHF (5V). The mixture was cooled to 0° C. and the mixture was purgedwith nitrogen gas. 1M Lithium triethylborohydride (1 eq) in THF wasadded at 0(±5°) C over a period of 1-2 hours. The mixture was stirred at0° C. for 30 minutes, warmed to 30° C. and stirred for 2 hours beforemethanol (10 V) was slowly added at 5° C. 10V 10% sodium hydroxide andthen 10V 10% hydrogen peroxide solution were added drop wise at 5° C.The mixture was extracted with ethyl acetate and the combined organiclayers were washed sequentially with 10% sodium metabisulfite solution,water in to reactor, 7% sodium bicarbonate solution and 10% sodiumchloride solution before being concentrated in vacuo. The product waswashed with heptane before being dried under vacuum at 50° C. to obtain2′-silyl cordycepin in 70-100% yield.

The 2′-silyl cordycepin, 2.5 equivalents imidazole and 0.15 equivalentsDMAP were dissolved in 5V DMF. The mixture was cooled to 15° C. before2.5 equivalents TBDMSCI were added portionwise. The reaction was stirredfor 4 hours at 30° C. before being cooled to 15° C. 10V water was addedand the mixture was extracted with ethyl acetate. The organic layerswere washed with 7% sodium bicarbonate solution, water and 5% sodiumchloride before being concentrated in vacuo.

The mixture was dissolved in 8V and 2V water was added before themixture was cooled to 0° C. 2.5 Eq. trifluoroacetic acid was added tothe reaction mixture at 0° C. over a period of 30-60 min. The mixturewas warmed to 10° C. and stirred for 4 to 6 hours at 10° C. Water wasadded and the mixture was extracted with ethyl acetate. The combinedorganic layers were washed with 7% sodium bicarbonate solution, water(twice) and 5% sodium chloride solution before being concentrated invacuo. The product was washed with heptane and dried under vacuum toprovide 5′-silyl cordycepin 21 in 40-70% yield.

5′-Silyl Cordycepin 21 to S_(p)-NUC-7738

5′-silyl cordycepin 21 was dissolved in 10 V THF and cooled to 0° C.2.0M t-BuMgCI (2.5 equivalents) was added and the mixture was stirredfor 15 min. The S_(p) isomer of compound 5 (2.5 eq) was dissolved in 5 VTHF and was added to the reaction at 0° C. The mixture was stirred at 0°C. for 15 min before being warmed to 25° C. and stirred for a further 2hours. The reaction was quenched into 10% ammonium chloride solution (10vol) and extracted with ethyl acetate. The combined organic layers werewashed with water and 10% brine solution before being concentrated invacuo.

The product was dissolved in THF (10V) before being cooled to 0° C. A 10V TFA and water (1:1) mixture was added to the reaction over a period of30 min before the mixture was stirred for 45 min, warmed to 30° C. andstirred for a further 16 h. The reaction was quenched in to 7% NaHCO₃solution (90 V) at 0° C. before being extracted with ethyl acetate. Thecombined organic layers were washed with water, 7% sodium bicarbonatesolution and 10% brine solution before being concentrated in vacuo.

The product was purified by column chromatography by using silica gel(100-200 mesh), the column was eluted by 2 to 10% MeOH in DCM to provideS_(p)-NUC-7738 in 40% yield. The HPLC purity of the product was 99.50%and Chiral HPLC showed the S_(p) isomer to be present in 99.90% and theR_(p) isomer to be present in 0.10%.

The same procedure can be carried out to provide R_(p)-NUC-7738.

Rp-NUC-7738:

¹H NMR (500 MHz, CDCl₃) δ_(H) 8.26 (s, 1H, H8), 8.22 (s, 1H, H2),7.37-7.25 (m, 7H, Ar), 7.22-7.12 (m, 3H, Ar), 6.01 (d, J=1.5 Hz, 1H,H1′), 5.12 (AB q, J_(AB)=12.0 Hz, Δδ_(AB)=0.04, 2H, CH₂Ph), 4.74-4.70(m, 1H, H2′), 4.69-4.62 (m, 1H, H4′), 4.44-4.38 (m, 1H, H5′), 4.28-4.21(m, 1H, H5′), 3.99-3.90 (m, 1H, CHCH₃ L-Ala), 2.35-2.27 (m, 1H, H3′),2.09-2.02 (m, 1H, H3′), 1.29 (d, J=7.0 Hz, 3H, CHCH₃ L-Ala).

³¹P NMR (202 MHz, CD₃OD) δ_(P) 3.91.

MS (ES⁺) m/z found 569.2 [M+H⁺], 591.2 [M+Na⁺], 1159.4 [2M+Na⁺]C₂₆H₂₉N₆O₇P required m/z 568.2 [M].

HPLC Reverse-phase HPLC (Varian Pursuit XRs 5 C18, 150×4.6 mm) elutingwith H₂O/CH₃CN from 90/10 to 0/100 in 30 minutes, F: 1 mL/min, λ=200 nm,shows one peak with t_(R) 14.02 min.

Sp-NUC-7738:

¹H NMR (500 MHz, CDCl₃) δ_(H) 8.24 (s, 1H, H8), 8.22 (s, 1H, H2),7.36-7.26 (m, 7H, Ar), 7.22-7.13 (m, 3H, Ar), 6.01 (d, J=1.5 Hz, 1H,H1′), 5.08 (AB q, J_(AB)=12.0 Hz, Δδ_(AB)=0.01, 2H, CH₂Ph), 4.70-4.67(m, 1H, H2′), 4.66-4.60 (m, 1H, H4′), 4.41-4.35 (m, 1H, H5′), 4.26-4.19(m, 1H, H5′), 4.02-3.94 (m, 1H, CHCH₃ L-Ala), 2.36-2.27 (m, 1H, H3′),2.08-2.01 (m, 1H, H3′), 1.34-1.30 (m, 3H, CHCH₃ L-Ala).

³¹P NMR (202 MHz, CD₃OD) δ_(P) 3.73. MS (ES⁺) m/z found 569.2 [M+H⁺],591.2 [M+Na⁺], 1159.4 [2M+Na⁺] C₂₆H₂₉N₆O₇P required m/z 568.2 [M].

HPLC Reverse-phase HPLC (Varian Pursuit XRs 5 C18, 150×4.6 mm) elutingwith H₂O/CH₃CN from 90/10 to 0/100 in 30 minutes, F: 1 mL/min, λ=200 nm,shows one peak with t_(R) 14.26 min.

The stereochemistry (R_(p) vs S_(p)) of the two NUC-7738 isomersdescribed above has been confirmed by conventional X-raycrystallographic analysis.

Example 10—Formation of NUC-9701

Dimethyl acetal 25 of 8-chloro-adenosine 24 can be made according to thefollowing scheme (also described in WO2017/207989).

Compound 25 can then be coupled with a compound of formula IIa using thecoupling process conditions described in Example 8. To form NUC-9701,dimethyl acetal can be removed using 1:1 TFA:water at 0° C. for 5 h.

Compound 25 (30 g; 1 equivalent) and the desired isomer of compound 12(58.08 g; 1.2 equivalents) were dissolved in 300 ml (10 V) of THF. Themixture was cooled to 0° C. before t-butyl magnesium chloride (76.8 mlof 2.0 M in THF; 1.75 equivalents) was added slowly, maintaining thetemperature at 0° C., and the mixture was stirred for 4 hours. 300 mL(10V) of 10% Ammonium chloride solution was added to the reactionmixture, maintaining the temperature at below 15° C. The mixture wasextracted with ethyl acetate and the combined organic layers were washedwith 7% sodium bicarbonate solution (twice), water (twice) and 20%sodium chloride before being dried with anhydrous sodium sulphate andfiltered. The ethyl acetate was removed in vacuo.

To the resultant product as added 600 ml (20V) of 60% formic acid inwater and the reaction was stirred for 65-70 h at 25° C. before ethylacetate (600 mL; 20V) was added slowly. 600 ml (20 V) of 20% sodiumchloride solution was added and the layers were separated. The aqueouslayer was extracted with ethyl acetate before 600 ml (20 V) 10% Ammoniasolution was added dropwise to the combined organic layers and thelayers were separated. The organic layer was washed with water (threetimes) and 20% sodium chloride solution, dried with 30 g (1 w/w) ofanhydrous sodium sulphate and filtered before being concentrated invacuo. The crude product was purified by column chromatography toprovide 20-45 g NUC-9701.

Sp-NUC-9701:

¹H-NMR (500 MHz; MeOD-d4): δ_(H) 8.07 (1H, d J=8.5 Hz, H-Napht), 8.05(1H, s, 2-H), 7.87 (1H, d J=8.5 Hz, H-Napht), 7.67 (1H, d J=8.5 Hz,H-Napht) 7.54-7.48 (2H, m, H-Napht), 7.41-7.30 (1H, m, H-Napht),7.36-7.33 (1H, m, H-Napht), 7.26-7.22 (5H, m, —CH₂Ph), 6.03 (1H, d J=5.0Hz, H-1′), 5.33 (1H, t J=5.0 Hz, H-2′), 5.01, 4.98 (AB, J_(AB)=12.3 Hz,CH₂Ph), 4.65 (1H t J=5.5 Hz, H-3′), 4.49-4.45 (1H, m, H_(a)-5′),4.41-4.36 (1H, m, H_(b)-5′), 4.22-4.20 (1H, m, H-4′), 3.94-3.90 (1H, m,—CHCH₃), 1.17 (1H, d J=7.0 Hz, CH₃).

³¹P NMR (202 MHz, MeOD-d4): δ_(p) 3.93 (1P, s).

Reverse-phase HPLC, eluting with H₂O/CH₃CN from 90/10 to 0/100 in 30min; 1 mL/min, λ=254 nm, showed a peak with t_(R)=16.43 min

Rp-NUC-9701:

¹H-NMR (500 MHz; MeOD-d4): δ_(H) 8.10 (1H, s, H-2), 8.08 (1H, d J=8.5Hz, H-Napht), 7.87 (1H, d J=8.5 Hz, H-Napht), 7.67 (1H, d J=8.5 Hz,H-Napht), 7.53-7.50 (1H, m, H-Napht), 7.48-7.44 (1H, m, H-Napht),7.40-7.38 (1H, m, H-Napht), 7.33-7.27 (6H, m, H-Napht and —CH₂Ph), 6.02(1H, d J=5.0 Hz, H-1′), 5.28 (1H, t J=5.0 Hz, H-2′), 5.04, 5.02 (AB,J_(AB)=12.2 Hz, CH₂Ph), 4.63 (1H t J=5.5 Hz, H-3′), 4.48-4.46 (1H, m,H_(a)-5′), 4.38-4.35 (1H, m, H_(b)-5′), 4.23-4.20 (1H, m, H-4′),4.05-4.01 (1H, m, —CHCH₃), 1.17 (1H, d J=7.0 Hz, CH₃).

³¹P NMR (202 MHz, MeOD-d4): δ_(P)3.83 (1P, s).

Reverse-phase HPLC, eluting with H₂O/CH₃CN from 90/10 to 0/100 in 30min; 1 mL/min, λ=254 nm, showed a peak with t_(R)=16.59 min

The stereochemistry (R_(p) vs S_(p)) of the two NUC-9701 isomersdescribed above has been assigned tentatively on the basis of comparisonof ³¹P chemical shift, 1H NMR spectra, and HPLC retention times withthose of other ProTides known in the literature.

What is claimed is:
 1. A process for the preparation of the (S_(p))-diastereoisomer of NUC-3373 (Formula Ia) having a diastereoisomeric purity of greater than 95%:

the process comprising steps a) to d) and, where P¹ is a protecting group, step e): a) suspending or dissolving the Y-diastereoisomer of the compound of Formula IIa, or a mixture of the Y- and X-diastereoisomers of the compound of Formula IIa, wherein R¹ is selected from the group consisting of halo group, trifluoromethyl, cyano and nitro and a is an integer from 1 to 5, in a solvent (S2), wherein S2 is a hydrocarbon or a mixture comprising a hydrocarbon:

b) treating the solution or suspension with a base (B2) to obtain the X-diastereoisomer having a diastereoisomeric purity of greater than 95%, wherein B2 is an organic amine base or an inorganic base; c) isolating the X-diastereoisomer of Formula IIa; d) reacting the X-diastereoisomer of Formula IIa with a compound of Formula Ma in presence of a base (B1) to provide a compound of Formula IVa having a diastereoisomeric purity of greater than 95%; wherein P¹ is independently selected from hydrogen and a protecting group:

and e) removing the protecting group P′ from the compound of formula IVa to provide NUC-3373 having a diastereoisomeric purity of greater than 95% wherein the X-diastereoisomer of the compound of Formula IIa is the (S_(p))-diastereoisomer and the Y-diastereoisomer of the compound of Formula IIa is the (R_(p))-diastereoisomer, except when the OPh(R¹)_(a) leaving group has lower priority assignment under the Cahn-Ingold-Prelog rules than the naphthyloxy group, in which case the X-diastereoisomer of the compound of Formula IIa is the (R_(p))-diastereoisomer and the Y-diastereoisomer of the compound of Formula IIa is the (S_(p))-diastereoisomer.
 2. The process of claim 1, wherein B1 is a Grignard reagent.
 3. (S_(p))-NUC-3373:

having a diastereoisomeric purity of greater than 95%.
 4. The process of claim 1 wherein the organic amine base B2 is selected from: N-alkylimidazole, pyridine, collidine, and 2,6-lutidine.
 5. The process of claims 1 wherein the organic amine base is a tertiary amine.
 6. The process of claim 5, wherein the organic amine base is a trialkylamine.
 7. The process of claim 6 wherein the organic amine base is triethylamine. 